PROJECT SUMMARY Small cell lung cancer (SCLC) remains a major challenge in public health because of its frequency, its lethality, and the paucity of convenient models for exploring its pathogenesis and potential therapeutic strategies. Pulmonary neuroendocrine cells (PNECs) are believed to be the putative precursor of SCLC. However, increasing evidence shows that PNECs contain sub-lineages varying in location, cell size, and physiological functions. In my previous research, I developed a novel experimental approach for studying the biology of PNECs - and the initiation of SCLC - by differentiating human embryonic stem cells (hESCs) into the lung lineage, and subsequently perturbing three tumor suppressor genes that are frequently altered in SCLC. By perturbing NOTCH signaling, the lung progenitor cells can be differentiated into PNECs that further undergo oncogenic transformation and form SCLC-like tumors in mice, when RB and P53 expression are reduced. Single cell RNA (scRNA) profiles demonstrated great similarity between the hESC-derived PNECs and the native PNECs in human and mouse lung. Of particular significance, scRNA analysis further revealed sub-lineages within the hESC-derived PNECs. Among them, one profile demonstrated significant similarity to the RNA profiles of early stage human SCLC tumors and SCLC cell lines. The above findings and recent studies by others, led me to further hypothesize that the PNEC sub-lineages have different oncogenic potential, and among them, one specific population serves as the dominant cell of origin of SCLC. I propose to use this model together with other methods such as scRNA transcriptomics, genetically engineering mouse models, to test this hypothesis and to study the origins of SCLC in several ways. First, I will identify the PNEC sub- lineages in normal human and mouse lung tissues that are similar to the ones in the hESC-derived PNECs. Alternatively, sub-lineages of PNECs in mouse lung will be characterized by scRNA profiling and new cell-fate markers identified from the mouse PNEC sub-lineages will be extrapolated to further delineate the heterogeneous populations in human PNECs. Next, I will purify the sub-populations of hESC-derived PNECs and test their transformative potential in culture and in immunocompromised mice by known oncogenic events in SCLC. Alternatively, the PNEC sub-lineages in mouse lung can be tested for their potential to form tumors by using conditional Rb1/Trp53 knockout mice. Through these studies, I expect to identify a specific sub-lineage of PNECs that are most sensitive to SCLC mutations and capable of transformation, which would implicate them as the cell of origin in SCLC. In the R00 phase, I propose to expand the research to explore mechanisms driving the lineage hierarchies of PNECs and their variant oncogenic potentials. These include studying inter-differentiation among the PNEC sub-lineages, the effects of NOTCH, SOX2 and other single pathways on PNEC fate determination, and using CRISPR screening to explore new molecular events important in maintenance of PNEC hierarchical patterns and the regulation of their specific oncogenic capacity. These studies are expected to not only advance our understanding of carcinogenesis of PNECs, but also provide new opportunities to diagnose, categorize, treat, and possibly even prevent this disease more effectively.